U.S. patent number 5,192,312 [Application Number 07/664,902] was granted by the patent office on 1993-03-09 for treated tissue for implantation and methods of treatment and use.
This patent grant is currently assigned to Colorado State University Research Foundation. Invention is credited to E. Christopher Orton.
United States Patent |
5,192,312 |
Orton |
March 9, 1993 |
Treated tissue for implantation and methods of treatment and
use
Abstract
Tissue which is suitable for transplant is treated with a growth
factor and cells which populate the tissue and native cells must be
removed, they cannot be "masked" reduce immunogenicity; this
increases the longevity of the tissue upon transplant. The
preferred growth factor is basic fibroblast growth factor, and the
preferred cells are fibroblasts. The tissue can be an allograft or
xenograft taken from a cow, pig or other mammal.
Inventors: |
Orton; E. Christopher (Fort
Collins, CO) |
Assignee: |
Colorado State University Research
Foundation (Fort Collins, CO)
|
Family
ID: |
24667918 |
Appl.
No.: |
07/664,902 |
Filed: |
March 5, 1991 |
Current U.S.
Class: |
600/36; 623/918;
427/2.24 |
Current CPC
Class: |
A61L
27/227 (20130101); A61L 27/54 (20130101); A61L
27/3625 (20130101); A61F 2/24 (20130101); A61L
27/3804 (20130101); A61F 2/0077 (20130101); A61L
27/38 (20130101); A61L 27/3695 (20130101); A61F
2/10 (20130101); Y10S 623/918 (20130101); A61L
2300/414 (20130101); Y10S 623/921 (20130101); A61F
2/2415 (20130101); A61L 2300/64 (20130101); A61F
2/062 (20130101) |
Current International
Class: |
A61F
2/00 (20060101); A61L 27/54 (20060101); A61L
27/00 (20060101); A61L 27/38 (20060101); A61F
2/24 (20060101); A61F 002/24 (); A61F 002/02 ();
A61F 002/54 (); A01N 001/02 () |
Field of
Search: |
;623/66,11,1,2
;427/2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
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|
|
0296475 |
|
Dec 1988 |
|
EP |
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0361957 |
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Apr 1990 |
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EP |
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WO86/02273 |
|
Apr 1986 |
|
WO |
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WO89/00198 |
|
Jan 1989 |
|
WO |
|
WO89/01286 |
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Feb 1989 |
|
WO |
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WO89/08117 |
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Sep 1989 |
|
WO |
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2136533 |
|
Sep 1984 |
|
GB |
|
Other References
O'Brien, et al., A Comparison of Aortic Valve Replacement with
Viable Cryopreserved and Fresh Allograft Valves, with a Note on
Chromosomal Studies, J. Thoracic & Cardiovascular Surg.
94:812-823 (1987). .
Slavkin, et al., Concepts of Epithelial-Mesenchymal Interactions
During Development: Tooth and Lung Organogenesis, J. Cellular
Bioch. 26:117-125 (1984). .
Manu-Tawiah and Martin, Peat Extract as a Carbon Source for the
Growth of Pleurotus ostreatus Mycelium, J. Sci. Food Agric.
47:243-247 (1989). .
Nichols, et al., Cytogenetic Evaluation of Human Endothelial Cell
Cultures, C. Cellular Phys. 132:453-462 (1987). .
Nanchahal, et al., Cultured Composite Skin Grafts: Biological Skin
Equivalents Permitting Massive Expansion, The Lancet 2 (1989)
22:191-193 (Jul. 22, 1989). .
Shiogama, et al., An Improved Cryopreservation Procedure for Human
Fetal Pancreas Tissues, Biological Abstracts, vol. 85, Abstract No.
46563 (1988). .
Shingh, et al., Binding of Tumor-Derived Angiogenic Factor to High
Affinity Receptor on Endothelial Cells, J. Cell Biology 103:299a,
Abstract No. 1107 (1986). .
Bell, et al., Living Tissue Formed in vitro and Accepted as
Skin-Equivalent Tissue of Full Thickness, Science 211:1052-1054
(1981). .
Weinberg and Bell, A Blood Vessel Model Constructed from Collagen
and Cultured Vascular Cells, Science 231:397-400 (1986). .
Kent, et al., Species Variation and the Success of Endothelial
Seeding, J. Vascular Surg. 9:271-276 (1989). .
Hoch et al., In vitro Endothelialization of an Aldehyde-Stabilized
Native Vessel, J. Surg. Res. 44:545-554 (1988). .
Noyes, Culture of Human Fetal Liver, Proc. Soc. Exp. Biol. Med.
144:245-248 (1973). .
Leapman, et al., Transplantation of Fetal Intestine: Survival and
Function in a Subcutaneous Location in Adult Animals, Ann. Surg.
179:109-114 (1974). .
Brockbank, Repopulation of Xenograft Heart Valves with Fibroblasts,
Small Business Innovation Research Grant Application No. PHS 90-2
(Submitted Aug. 15, 1990)..
|
Primary Examiner: Isabella; David
Assistant Examiner: Nguyen; Dinh X.
Attorney, Agent or Firm: Kenyon & Kenyon
Claims
I claim:
1. An implantable human heart valve treated with growth factor
effective on fibroblast cells and populated with fibroblast cells
in an amount and for a time period effective for rendering the
heart valve substantially non-immunogenic upon implant into a
mammal.
2. An implantable non-human mammalian heart valve treated with
growth factor effective on fibroblast cells and populated with
fibroblast cells in an amount and for a time period effective for
rendering the heart valve substantially non-immunogenic upon
implant into a mammal.
3. The substrate of claim 2 wherein the non-human mammalian heart
valve is porcine or bovine in origin.
4. The heart valve of claims 1, 2 or 3 wherein the fibroblast cells
include autologous cells.
5. The heart valve of claims 1, 2 or 3 wherein the fibroblast cells
include allogeneic cells.
6. The heart valve of claims 1, 2 or 3 wherein the heart valve is
sterilized prior to treatment with growth factor.
7. The heart valve of claim 1 wherein the growth factor used to
treat the heart valve includes basic fibroblast growth factor.
8. The heart valve of claim 1 wherein the growth factor used to
treat the heart valve includes acidic fibroblast growth factor.
9. The heart valve of claim 2 wherein the growth factor used to
treat the heart valve includes basic fibroblast growth factor.
10. The heart valve of claim 2 wherein the growth factor used to
treat the heart valve includes acidic fibroblast growth factor.
11. A method of reducing the immunogenicity or improving the
longevity of an implantable mammalian heart valve comprising:
treating the heart valve with growth factor effective on fibroblast
cells, and
populating the heart valve with fibroblasts in an amount effective
for reducing the immunogenicity of the heart valve upon implant
into a patient.
12. The method claim 11 wherein the growth factor used to treat the
heart valve includes basic fibroblast growth factor.
13. The method claim 11 wherein the growth factor used to treat the
heart valve includes acidic fibroblast growth factor.
14. The method claim 11 wherein the heart valve is exposed to
radiation prior to treatment with the growth factor.
15. The method claim 11 wherein the heart valve is sterilized prior
to treatment with the growth factor.
16. The method claim 11 wherein the fibroblast cells include
autologous cells.
17. The method claim 11 wherein the fibroblast cells include
allogeneic cells.
18. A method of reducing transplant tissue rejection of an
implantable mammalian heart valve comprising:
treating the heart valve with growth factor effective on fibroblast
cells, and
populating the treated heart valve with fibroblasts to reduce
tissue rejection upon transplant into a patient.
19. The method claim 18 wherein the growth factor used to treat the
heart valve includes basic fibroblast growth factor.
20. The method claim 18 wherein the growth factor used to treat the
heart valve includes acidic fibroblast growth factor.
21. The method claim 18 wherein the heart valve is sterilized prior
to treatment with the growth factor.
22. The method claim 18 wherein the heart valve is exposed to
radiation prior to treatment with the growth factor.
23. The method claim 18 wherein the fibroblast cells include
autologous cells.
24. The method claim 18 wherein the fibroblast cells include
allogeneic cells.
Description
During the last 20 years, allograft heart valve transplantation in
the United States has increased from about 50 to 2,000 per annum.
Because of the increase in demand, particularly in pediatric cases,
utilization of allograft heart valves is now limited by the supply
of donated human hearts.
The invention described herein relates to transplantable tissue,
such as heart valves, which is treated to reduce potentially
untoward reactions to the tissue which would otherwise result upon
transplant. Implantable tissue has in the past been taken from
patients and reimplanted into the same patient in a different site,
such as with burn victims who require skin grafts and coronary
bypass patients who require coronary arterial replacement using
sections of saphenous veins.
Similarly, organs such as kidneys have been transplanted
allogeneically from one sibling to another in an effort to minimize
immunologically mediated reactions by the transplant recipient,
which would result in organ rejection. These patients, as well as
patients receiving transplant organs from donors other than
siblings, are frequently administered drugs to suppress the immune
system. While the immunological response to transplant tissue may
be suppressed through the use of immunosuppressant drugs to
minimize tissue rejection, immunosuppressant therapy is general in
nature. Hence, immunosuppressant drugs also tend to suppress the
immune response, which reduces the transplant patient's ability to
combat infection.
The supply and ready availability of transplantable organs and
graft tissue has been far outdistanced by the demand for such
tissue over the past several years, and there is a long-felt need
for an increase in the supply of such organs and tissue. This need
remains to an extent unfilled, even taking into account the various
synthetic tissues and mechanical organs which are presently
available.
Bioprosthetic grafts are typically superior to mechanical
prosthetic devices for various reasons. For example, mechanical
heart valves are typically more prone to cause thromboembolism than
bioprosthetic grafts. Moreover, mechanical equipment failures
typically occur suddenly and without warning, resulting in
emergency situations requiring surgical intervention and
replacement of the artificial prosthetic device. Bioprosthetic
heart valve grafts do not typically fail suddenly when a problem
occurs. Rather, if there is a secondary valve failure, the valve
tends to wear out gradually over time. This gives the patient and
treating physician some advance warning that a graft prosthesis
failure is likely to occur.
The invention described herein relates to xenogeneic or allogeneic
tissues made suitable for transplant into a patient by replacing
native cells within the tissue with autogenous or allogeneic cells.
These modified grafts combine the advantages of bioprosthetic
valves with immunological tolerance on the part of the recipient
and the ability to maintain and repair the extracellular
matrix.
There have been attempts at producing artificial tissues and organs
in the past with varying degrees of success.
Steinberger, U.S. Pat. No. 4,407,787, relates to a dressing
comprised of collagen and a resorbable biopolymer. The dressing is
tissue-agglutinable, such that the dressing adheres to tissue and
causes hemostasis.
Caplan, et al., U.S. Pat. No. 4,609,551, relates to a process for
stimulating bone and cartilage growth, utilizing a soluble bone
protein. The bone protein is combined with cells such as
fibroblasts, and the mixture may be injected into the site of a
joint cavity articular surface defect. Alternatively the bone
protein and cells may be implanted in a fibrin clot. The
fibroblasts differentiate to form replacement cartilage tissue.
Nevo, et al., U.S. Pat. No. 4,642,120, relates to a gel-type
composition for repairing bone and cartilage defects. The gel
contains mesenchymal cells which differenciate into cartilage cells
through the influence of chondrogenic inducing factor in
combination with fibrinogen, antiprotease and thrombin.
Bell, U.S. Pat. No. 4,485,096, relates to a tissue equivalent for
treatment of burns or skin wounds and to fabricated prostheses. A
hydrated collagen lattice is contracted with a contractile agent,
e.g., fibroblasts or blood platelets, to create a collagen lattice
which may then be populated with keratinocytes, thus forming a skin
equivalent. Alternatively, glandular cells, such as pancreatic beta
cells, or hepatocytes can be grown on the collagen lattice to
produce a pancreas or liver tissue "equivalent". Bone equivalents
can also be formed from the contracted collagen matrix described
above in combination with demineralized bone powder.
Bell, U.S. Pat. No. 4,539,716, similarly relates to synthesized
equivalents for blood vessels and glandular tissue. A contractile
agent is used to contract the collagen lattice axially around an
inner core. Additional layers containing capillary beds, blood
vessels and glandular structures are then constructed.
Bell, U.S. Pat. No. 4,546,500, relates to the fabrication of blood
vessels and glandular tissues utilizing a collagen lattice
contracted axially around an inner core and combined with a plastic
mesh sleeve. The plastic sleeve is sandwiched between layers of the
matrix to reinforce the structure.
Bell, et al., U.S. Pat. No. 4,835,102, relates generally to tissue
equivalent test systems, and includes tissue equivalents for
epithelial, connective, cartilage, bone, blood, organs and
glandular tissues as well as blood vessels. The tissue equivalent
is composed of cultured cells which are derived from the endogenous
tissue and incorporated into a collagen lattice.
Bell, et al., PCT Application WO 86/02273 published Apr. 24, 1986,
relates to methods of forming living tissue equivalents, which
utilize a collagen matrix contracted to form a lattice in a
nutrient medium. The initially acidic collagen system is
precipitated by raising the pH sufficiently to induce
fibrillogenesis and the formation of a gel matrix containing
cells.
Bell, et al., European Patent Application No. 89309972.1 relates to
tissue equivalents which have cell types differentiated from
progenitor cells without exogenous chemical induction. The tissue
equivalent is in the form of a tissue precusor mixture which is
non-gelled, and the mixture is injected into the host. The mixture
gels and is space filling upon injection into the appropriate site.
The cells must exhibit the ability to differentiate without
exogenous chemical induction for the tissue equivalent to be
effective.
Shing, Y. et al. Cell Biology, Vol. 103, No. 5, Pt. 2 Abstract No.
1107, page 299a (1986) relates to a chondrosarcoma derived growth
factor which is angiogenic in vivo. The chondrosarcoma growth
factor is used to stimulate endothelial cell proliferation in
vitro.
Bell, et al. Science Vol. 211, pp 1052-1054 (1981) relates to
skin-equivalent grafts treated with a contractile agent to form a
collagen lattice. The lattice is seeded with epidermal cells. The
lattice allegedly permits vascularization of the graft.
Weinberg, C. B., et al., Science Vol. 231: 397-400 (1986), relates
to a blood vessel model containing collagen and cultured vascular
cells.
Kent, K. C. et al., J. Vascular Surg. Vol. 9, No. 2, pp 271 to 276
(1989), relates to endothelial seeding of vascular grafts in dogs,
and the patency of the luminal monolayer. Endothelial cells
harvested from bovine aorta, canine external jugular vein and human
saphenous vein are compared.
Hoch, J. et al. J. Surg. Res. vol. 44, No. 5, pp. 545 to 554,
relates to the use of Dacron and polytetrafluoroethylene polymeric
grafts, as well as bovine carotid artery heterografts which were
compared in vitro to determine the extent of endothelial cell
adherence.
Noyes, W. F. Proc. Soc. Exp. Biol. Med. Vol. 144, No. 1 pp. 245-248
(1973) relates to human liver cell cultures which utilize collagen
as a substrate. Gel-foam sponge is also used as a substrate.
The present invention relates to a transplantable or implantable
xenogeneic or allogeneic tissue having immunogenic sites which if
untreated, would ordinarily induce an immune system response in the
patient, ultimately leading to transplant rejection. Similarly, a
method is described of rendering the transplantable tissue
substantially non-immunogenic by replacing native cells with
allogeneic or autologous cells, reducing the recognition of a
transplanted graft as a foreign substance without generally
suppressing the patient's immune system.
In particular, the present invention relates to transplantable
tissue which can be treated in accordance with the methods
described herein to reduce or prevent untoward immune system
reactions which the recipient may experience in response to the
graft, which in turn minimizes transplant rejection. Hence, one
object of the present invention is to reduce patient rejection of
transplanted tissue.
A further object of the present invention is to increase the supply
of transplantable tissue by treating grafts to render them suitable
for transplant into human patients in need of such treatment.
A further object of the present invention is to facilitate the use
of animal donors that can supply xenograft donor tissue in
virtually unlimited quantities. The donor tissue can be
transplanted into human recipients after the tissue has been
treated in accordance with the methods described herein.
Additional objects of the present invention will be apparent to
those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
The invention described herein includes a transplantable
bioprosthetic graft tissue which is treated prior to transplant
with a growth factor and then exposed to cells which are attracted
into the tissue and proliferate in response to the growth factor
and populate the transplantable tissue. Replacement of cells
effectively reduces immune responses to the tissue, thus improving
the effective life of the graft and reducing the frequency,
incidence and severity of transplant rejection.
The invention further addresses a method of treating xenogeneic
transplantable tissue which comprises exposing the tissue to a
growth factor and then culturing the graft tissue with cells which
migrate and proliferate in response to the growth factor, thus
populating the tissue with the cells to enhance the effective life
of the tissue upon transplant, and reduce any immunologically
mediated adverse effects which the graft recipient otherwise
experiences in response to the xenogeneic tissue upon
transplantion.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a photomicrograph of control tissue not exposed to growth
factor, and
FIG. 2 is a photomicrograph of tissue exposed to basic fibroblast
growth factor ("bFGF") (2500 ng/ml) and incubated with fibroblasts
for 10 days.
DETAILED DESCRIPTION
The terms "tissue", "organ" and "organ part" are used in the
general sense herein to mean any transplantable or implantable
tissue, organ or organ part, the survivability of which is improved
by the methods described herein upon implantation. In particular,
the overall durability and longevity of the implant are improved,
and host-immune system mediated responses, e.g., graft rejection,
are reduced in severity as well as in frequency, and may be
eliminated altogether.
The terms "transplant" and "implant" are used interchangably to
refer to tissue or cells (xenogeneic or allogeneic) which may be
introduced into the body of a patient to replace or supplement the
structure or function of the endogenous tissue.
The term "autologous" refers to tissue or cells which originate
with or are derived from the recipient, whereas the terms
"allogeneic" and "allograft" refer to cells and tissue which
originate with or are derived from a donor of the same species as
the recipient. The terms "xenogeneic" and "xenograft" refer to
cells or tissue which originates with or is derived from a specie
other than that of the recipient.
The invention described herein is particularly useful for
bioprosthetic xenografts in which the major structural component is
connective tissue matrix. Examples of such grafts include
bioprosthetic heart valves, blood vessels, ligaments and
tendons.
Hence, a preferred aspect of the invention encompasses a xenograft
treated with a growth factor and incubated with cells that migrate
and proliferate in response to the growth factor, thus populating
the xenograft, said replacement of cells being effective for
reducing allergic complications upon transplant when compared to
untreated xenografts.
Upon treatment of the xenograft with growth factor according to the
methods described herein, and upon population of the xenograft with
allogeneic or autogenous cells that improve the viability of the
xenograft after transplant and reduce any immune response to the
xenograft, there is a reduced tendency for thromboemboli to occur,
particularly when compared to mechanical heart valves. This results
in increased implant longevity, decreased or slowed degeneration of
the implant, and decreased adverse immune reactions which otherwise
may result in host rejection.
The preferred growth factor for use herein is fibroblast growth
factor, in particular, basic fibroblast growth factor ("bFGF").
When used to treat xenograft implants, such as heart valves, the
graft may be initially exposed to a buffered nutrient medium, and
then immersed in a solution containing bFGF. Optionally the graft
may be sterilized and rendered acellular using an effective dose of
radiation or a cytotoxic solution prior to treatment with bFGF.
The concentration of growth factor used to treat the xenograft
typically ranges from about 100 ng/ml to 10 mg/ml with a growth
factor concentration for bFGF of about 2.5 mcg/ml being most
preferred.
The graft is bathed in the solution containing growth factor for a
time period which is effective for causing cells which migrate and
proliferate in response to the growth factor to adhere to and
penetrate the surface of the xenograft. This, in effect, causes the
cells to populate the xenograft.
To populate (or repopulate) the graft with cells, the graft may be
washed, immersed in a growth factor containing solution, and then
placed into a suitable buffered medium containing the cells which
migrate and proliferate in response to the growth factor, thus
populating the graft tissue with cells. The graft and cells are
cultured together at a temperature and for a time period which are
effective for causing the cells to populate and adhere to the
graft.
Culture times range from about 3 to 21 days. Culture times may be
reduced somewhat by increasing the initial concentration of
cells.
When fibroblasts are used as the graft-populating cells, the graft
may typically be immersed in Dulbecco's Modified Eagle medium with
5% serum. The graft is cultured with a primary fibroblast culture
for about three days. Additionally the graft may be secured on the
culture plate and incubated at about 37.degree. C. in a humidified
atmosphere, until the graft has been populated with fibroblasts,
e.g. 5% CO.sub.2 /95% air. Incubation is considered complete when
the fibroblasts have populated the graft in such a manner that the
graft appears histologically similar to a fresh graft. (e.g., a
normal cell distribution).
Essentially any buffered physiological salt solution containing
protein carriers can be employed.
Preferred buffers for use with the growth factor include sodium
phosphate/glycerin/bovine serum albumin ("BSA"). These buffers
typically are used to provide a physiologically acceptable pH, such
as about 7.0 to 7.6.
The cells which are used to populate the graft can be varied within
wide limits, and different types of cells can be used in different
circumstances, depending upon the site and size of the transplant,
the nature of the tissue to be replaced, the allergic sensitivity
(or hypersensitivity) of the patient and other factors.
The graft may be sterilized prior to treatment with the growth
factor, or treated to kill off the endogenous cells in the graft
prior to treatment with growth factor and subsequent graft
population. This may reduce the likelihood of microorganismal
contamination as well as the immunogenicity of the graft prior to
graft population and implantation. A preferred method for
sterilizing grafts prior to population utilizes radiation exposure,
e.g., x-rays in lethally effective doses. Alternatively,
antibiotics, antibacterials and cytotoxic agents in normally
effective doses may be used.
A preferred aspect of the invention involves the use of autogenous
cells in the process described herein. In this instance, a tissue
sample is taken from the patient prior to transplant surgery. The
tissue is treated in accordance with the methods described herein
to produce fibroblasts or other cells which are then used to
repopulate the graft. By immersing the graft in growth factor and a
culture of autogenous cells, and by populating the graft with cells
derived from the resected tissue taken from the patient, an adverse
immune system response and ultimately graft rejection can be
minimized or avoided.
The cell source tissue can be selected to match the tissue to be
transplanted. For example, if a blood vessel is to be transplanted,
cells can be taken from a patient, healthy blood vessel and used as
the source of cells for graft population. In this fashion, the
healthy graft can be very closely matched to the patient's diseased
tissue.
This aspect of the invention is particularly useful when the
transplant patient is highly allergic, or if the tissue is highly
immunogenic, such as with respect to transplantable blood
vessels.
Alternatively, cell lines can be used to repopulate the graft which
are substantially non-immunogenic. Cells which elicit no more than
a weak allergic response are used to populate the graft prior to
transplant.
Method for isolation of fibroblasts
The tissue, for example skin or heart valve leaflet, is cut into 1
mm.sup.3 pieces using a sterile dissection technique. Groups of 10
pieces are then placed in 35 cm.sup.2 tissue culture dishes with
approximately 1 ml of culture medium (DMEM+10% FCS). It is
important that the pieces of tissue remain attached to the plastic
surface of the culture dish; if the tissue detaches, the amount of
culture medium should be reduced. Incubate for 1 week at 37.degree.
C. in a humidified culture incubator. After 1 week of incubation,
each piece of tissue is surrounded by a dense outgrowth of
fibroblasts. Epithelial cells may also be present but are lost
during subsequent cell culturing. The fibroblasts are removed with
a plastic scraper or by collagenase digestion after rinsing the
cells with a calcium and magnesium-free buffered salt solution, and
placed in larger cell culture vessels with fresh culture medium.
The cell cultures can be expanded in this manner. The contents of
one flask can be divided and placed into three larger vessels, and
this process can be repeated about once a week for at least 10
weeks. These flasks of fibroblasts are then utilized as a cell
source. Cells obtained in this manner are preferable to
commercially available cell lines, because most cell lines are
genetically modified and are no longer responsive in a normal
manner to growth regulators (such as FGF).
The fibroblasts can be either immunologically matched allogeneic
cells, such that the recipient does not recognize them as foreign,
or autologous cells, in which case the donor and recipient are the
same individual.
Results
A study was performed using canine leaflets and bovine fibroblasts.
Mitral valve leaflets were aseptically harvested from a dog cadaver
shortly after it was killed. The leaflets were divided into
sections and placed in petri dishes containing 5 ml NaH.sub.2
PO.sub.4 /glycerin/BSA buffer. The leaflets were irradiated with
4,000 cGy of 6 MV x-rays to kill the donor cells. The leaflets were
then placed in HBSS (Hanks Balance Salt Solution) with 0.25%
trypsin for 10 minutes to remove any residual endothelial cells.
The trypsin was inactivated by adding cold culture medium with 5%
serum. The leaflets were washed and placed in NaH.sub.2 PO.sub.4
/glycerin/BSA buffer. Human recombinant bFGF was added to the
NaH.sub.2 PO.sub.4 /glycerin/BSA buffer in the following
concentrations: 0, 50, 500, and 2,500 ng/ml and incubated for 4.5
hours.
The stock solution of bFGF was prepared with sodium heparin added
in a 3:1 bFGF:heparin (w/w) ratio. Aliquots and the bFGF stock
solution were stored at -70.degree. C.
After incubation, the leaflets were washed in phosphate buffered
saline and placed in DMEM (with non-essential amino acids and
penicillin/streptomycin) with 0.5% fetal calf serum ("FCS"). Bovine
fibroblasts, which had previously been obtained from calf aorta by
standard explant techniques were added to the heart valve leaflets
at 2.times.10.sup.4 cells/ml. The heart valve leaflets were then
secured to the bottom of the plate with small weights and incubated
for 10 days at 37.degree. C. in a humidified 5% CO.sub.2 and 95%
air environment.
Following incubation, valve sections were placed in formalin for
histopathological analysis. The analysis demonstrated that there
was a bFGF dose-dependent increase in fibroblast ingrowth into the
heart valve leaflets. For example representative micrographs in
FIG. 1 show the control leaflets not exposed to bFGF were
essentially acellular, whereas leaflets exposed to 2500 ng/ml bFGF
shown in FIG. 2 were well populated with cells. These results
demonstrate that fibroblasts will populate an irradiated
FGF-treated xenograft.
The description contained herein contains the preferred embodiments
of the invention. However, numerous alternative embodiments are
contemplated as falling within the scope of the invention.
* * * * *